11alpha-hydroxy testosterones



Patented Aug. 17, 1954 Ila-HYDROXY TESTOSTERONES Herbert 0. Murray, Hickory Corners, and Diirey H. Peterson, Kalamazoo, Mich assignors to The Upjohn Company, Kalamazoo, Mich., a corporation of Michigan OFFICE No Drawing. Application January 15, 1953, Serial N0. 331,491

Claims. (Cl. 260--397.45)

This invention relates to steroids and more particularly to certain testosterone derivatives namely 11m hydroxytestosterone, 11a hy droxy l0 normethyltestosterone and their esters represented by the following formula:

CHa

is a continuation-in-part, S. N. 296,728, now

abandoned, and 296,729, filed July 1, 1952; and 278,122 and 278,123, filed March 22, 1952, now both abandoned, which are continuations-in-part of 272,944, filed February 23, 1952, now Patent 2,602,769, issued July 8, 1952, and 180,496, filed August 19, 1950, now abandoned, the novel compounds of the present invention may be prepared from testosterone or'lo-normethyltestosterone by the oxygenating action of a culture of fungus and the resulting oxygenated products may be esterifled.

Esterification may be accomplished by admixing lla-hydroxytestosterone or 11a-hydroxy-l0- normethyltestosterone with an acylating agent such as, for example, ketone, a ketene of a selected acid, an acid, acid chloride or acid anhydride, or other known acylating agent, usually in asolvent such as, for example, pyridine or the like, or an inert solvent, including solvents like benzene, toluene, ether, and the like, for example, and heated at a temperature between about zero degrees centigrade and the boiling point of the reaction mixture usually about room temperature, for a period between about a half hour and about 96 hours. The time of reaction as well as the temperature at which the reaction is carried out, the acylating. agent, and the ratio of reactants may be varied. The reaction mixture is suitably poured into ice or cold water, the product col- 2 lected in an appropriate solvent which is thereafter washed with successive portions of a mildly basic solution and water to obtain a solution of the product which is essentially neutral.

In some instances, the product may crystallize from the reaction mixture, in which case it may be advantageous to separate the product by filtration or other means, wash with water, and thereafter purify by conventional means, such as, for example, by recrystallization from a suitable solvent or by chromatographic purification, as deemed necessary.

The thus-described acylation process, and as illustrated in more detail in the examples following in this specification, produces both the monoesters and the (ii-esters, although in difierent proportions, depending upon the proportions of acylating agent to l1oc-I'lYdIOXYtGStOStGI'OllG or llwhydroxy l0 normethyltestosterone. Usingapproximately one equivalent of acylating agent to steroid produces predominantly the monoacylated product, whereas with about two or more equivalents of acylating agent to steroid, the predominant product is the diacylat'ed product.

The following examples are illustrative of the process and products of the present invention, but are not to be construed as limiting.

Emamp le 1 .-1 1 a hydroxytesws terone A medium was preparedof twenty grams of Edamine enzymatic digest of lactalbumin, three grams of corn steep liquor and fifty grams of technical dextrose diluted to one liter with tap water and adjusted to a pH of 4.3 to 4.5. Three liters of this sterilized medium was inoculated with Rhizopus nigricans minus strain, American Type Culture Collection Number 6227b, and incubated for 24 hours at a temperature of 28 degrees centigrade using a rate of aeration and stirring such that the oxygen uptake Was 6.3 to 7 millimoles per hour per liter of NazSOs according to the method of Cooper, Fernstrom and Miller,v Ind. Eng. chem, 36, 504 (1944). To this medium containing a, 24-hour growth of Rhizopus nigric ans minus strain was added 1.5 grams or testosterone in thirty milliliters of absolute ethanol to provide a suspension of the steroid in the culture. After an additional 24-hour period of incubation under the same conditions of temperature and aeration, the beer and mycelium were extracted, The mycelium was filtered, washed twice, each time with a volume of acetone approximately equal to the volume of the mycelium and extracted twice, each time with 2. volume of methylene chloride approximately equal to the'volume of the mycelium. The acetone and methylene chloride extracts including solvent were added to the beer filtrate. The mixed extracts and beer filtrate were extracted successively with two one-half by volume portions of methylene chloride and then with two one-fourth by volume portions of methylene chloride. The combined methylene chloride extracts were washed with two one-tenth by volume portions. of a two percent aqueous solution of sodium bicarbonate and then with two one-tenth by volume portions of water. After drying the methylene chloride with about three to five grams of anhydrous sodium sulfate per liter of solvent and filtering, the solvent was removed by distillation. The extractives obtained upon evaporation of the methylene chloride solvent weighed 2.5492 grams.

Partial decolorization of the crude extractives was effected by taking them up with 35 milliliters of methylene chloride, adding three grams of Magnesol synthetic magnesium silicate and filtering. Upon evaporation of the solvent, the residue was taken up in an excess of acetone, mixed with 0.2 gram of Celite No. 545 diatomaceous earth, filtered, and evaporated to dryness. This residue was dissolved in 150 milliliters of benzene and fractionated over 75 grams of alumina (washed with hydrochloric acid and dried at 120 degrees centigrade) with 150-milliliter portions of solvent fractions as in the table.

Table Eluate Solids Fraction Solvent Milligrams 1,2 benzene plus 10 percent ether 3,4 benzene plus 50 percent ether 57. ether ether plus 5 percent chl0roform etheri plus percent ch1oroform o ether plus 50 percent chloroform chloroform o chloroform plus 10 percent acetone chloroform plus 50 percent acetone. ecetone acetoneplus 5 percent methanol acetone-methanol, increased to 100 percent methanol.

Total.

Fractions 23 to 30, weighing 378 milligrams, freed of solvent, were dissolved in acetone, concentrated to ten milliliters, filtered to separate a small amount of flocculent precipitate, and evaporated to dryness. The residue was dissolved in fifteen milliliters of ethyl acetate, concentrated over a steam-bath to five milliliters, and cooled to crystallize the product. The crystals (409-SHE- 41B-1) were separated by filtration and washed three times with one-milliliter portions of ethyl acetate.

The mother liquor fraction identified as 409- SHE41B-l M. L. was combined with subsequently obtained fraction 41B2 M. L., as described below, and evaporated to crystallization. The crystals were Washed with cold ethyl acetate yielding sample 41B3 weighing 40.5 milligrams and melting at 177 to 178 degrees centigrade.

Crystals 409-SHE-4lB-l, obtained concomitantly with the above mother liquor fraction, weighing 319.9 milligrams, were taken up in methylene chloride, Magnesol was added to decolorize, the suspension was filtered and the filtrate was evaporated. The residue was redissolved in two milliliters of ethyl acetate and crystallized therefrom yielding 198.7 milligrams of crystals (409-SHE-41B-2) melting at 179 to 182 degrees centigrade and mother liquor fraction 4113-2 M. L. which mother liquor fraction is utilized as indicated in the preceding paragraph. These crystals were taken up in seven milliliters of ethyl acetate, concentrated to four milliliters and recrystallized, yielding 169.7 milligrams of llwhydroxytestosterone, softening at 178 degrees Centigrade, melting at 181 to 181.5 degrees centigrade and having an optical rotation of plus 93 degrees (chloroform). Infrared spectrum shows the presence of an additional hydroxyl on testosterone.

AnaZysis.-Calculated for 0191-12703: C, 74.97; H, 9.27. Found: C, 74.69; H, 9.26.

Example 2A.1 1 a-hydrory-l O-normethyltestosterone A medium was prepared of 0.5 gram of soy flour, two grams of dextrose, and 0.5 gram each of debittered brewers yeast extract, sodium chloride, and potassium dihydrogen phosphate diluted with tap water to milliliters and adjusted with hydrochloric acid to a pH of 4.5. This sterilized medium was inoculated with spores of Rhieopas reflerus ATCC 1225, from a malt agar slant, and

incubated for 24 hours at a temperature of 28 degrees centigrade using agitation and aeration corresponding to an oxygen uptake of eight millimoles per hour per liter of NazSOs according to Cooper, Fernstrom and Miller, Ind. Eng. Chem, 36, 504 (1944). To this medium, containing a 24-hour growth of Rhieopus reflercus, was added twenty miligrarns of 10-normethyltestosterone dissolved in one milliliter of acetone to produce a suspension of the steroid in the culture. After an additional 24-hour period of incubation under the same conditions of temperature and aeration, the beer and mycelium were extracted as in Example l. The concentrated extract was subjected to separation by paper chromatography using a propylene glycol-toluene system to yield llc-hydroxy 10 normethyltestosterone and 6/3- hydroxy-10-normethyltestosterone.

Example 2B.-1 1 a-hydroxy-l O-normethyltestostcro'ne Otherwise like Example 2A, similar results were produced by fermentation of Rhieopus refleosus in a medium consisting of two grams of corn steep liquor, two grams of dextrin, 0.10 gram of potassium dihydrogen phosphate, 0.2 gram of sodium nitrate, 0.5 gram of magnesium sulfate heptahydrate, 0.02 gram of potassium chloride, 0.001 gram of ferrous sulfate heptahydrate, and 0.25 gram sodium acetate diluted with tap water to 100 milliliters and adjusted with concentrated sodium hydroxide to a pH of 7.0.

Example 2C .-1 1 a-hydroxy-l O-normethyltes tosterone In the same manner as described in Example 1, l1uhydroxy-10-normethyltestosterone is prepared from lO-normethyltestosterone [Birch, J. Chem. Soc. (London), 1950, 367] by subjecting the latter steroid to the oxygenating activity of Rhizopas nigricans minus strain, American Type Culture Collection Number 62271). The resulting oxygenated steroid is isolated from the fermentation mixture in the same manner as described in Example 1 and purified 11a-hydroxy-10- normethyltestosterone is obtained by crystallization of the reaction product from ethyl acetate.

Example 3.-1la-acetoxytestosterone acetate To fifty milligrams (0.164 millimole) of 11a- 5. hydroxytestosterone dissolved inone milliliter. of barium oxide dried pyridine was added one milliliter (0.452 millimole) of a solutionxofe 0.462

milliliter of redistilled acetic vanhydride-diluted to ten milliliters with dry pyridine andthe. re-I action mixture maintained. at room temperafour one-milliliter portions of' ice water and;

thereafter dried in vacuumat sixty degree centirade. The dry product weighed 3.4 milligrams and melted at 187 to 195 degrees centigrade. In-1 frared sp ctrum analysis confirmed the theoretical structure. The product was dissolved in 1.5.

milliliters of methanol, the resulting solution filtered and thereafter diluted with 0.5 milliliter of water whereafter. there was precipitated crystals which were filtered, washed with three one-milliliter portions of ice water and thereafter dried at seventy degrees centigrade. The 110cacetoxytestosterone acetate thus produced weighing 22 milligrams melted at 201 to 203 degrees centigrade and had an M1 of plus 56 degrees (1.087 in chloroform).

Analysis-Calculated for CzaHszOs:

. C, 70.92; H, 8.52 C, 71.19; H, 8.46 C, 71.22; H, 8.36

Example 4.11a-form'yloxytestosterone formate In the same manner as Example 3, 110;-

Found:

formyloxytestosterone formate is prepared by reacting lla-hydroxytestosterone with more than about two equivalents of formic acid.

Example 5.-1Ia-propionyloxytestosterone propionate In the same manner as Example 3, 11ozpropionyloxytestosterone propionate is prepared by reacting lla-hydroxytestosterone with more than about two equivalents of propionic anhydride in pyridine.

Example 6.-11 a- (li-cyclopentyl) propz'onyloxytestosterone e-cyclopentylpropionate Example 7.11a-benzoxytestosterone benzoate In the same manner as Example 3, lla-benzoxytestosterone benzoate is prepared by reacting lla-hydroxytestosterone with more than about two molar equivalents of benzoyl chloride in pyridine.

Example 8.-.11a-acetoxy-lO-normethyltestostero'ne acetate In the same manner as Example 3, lla-acetoxylfl-normethyltestosterone acetate is prepared by reacting 1lot-hydroxy-lo-normethyltestosterone with more than about two equivalents of acetic anhydride in pyridine.

Example 9.1 1 a-formyloxy-i O-normethyltestosterone formate In I the same manner as Example 3, 11ozformyloxy-lo-normethyltestosterone formate is prepared by reacting 1la-hydroxy-m-normethyltestosterone with more than about two equivalents offormic acid.

Example 10.1la-propionyloxy-l0-norme1ihyh testosterone propz'onate In the same manner as Example 3, Ila-propionyloxy-IO-normethyltestosterone propionate is prepared by reacting lla-hydroxy-lo-normethyltestosterone with more than about two equivalents of propionic anhydride in pyridine.

Example 11 .-1 1 a (/8 cyclopentyl) propionloxy- 10 normethyltestosterone p cyclopentylpropionate In the same manner as Example 3, Ila-(pcyclopentyllpropionyloxy l0 normethyltestosterone ,B-cyclopentylpropionate is prepared by reacting 110a hydroxy 10 normethyltestosterone with more than about two equivalents of ,B-oyclopentylpropionyl chloride in pyridine.

Example 12,-11a-benzoxy 1 0-noxm6thyltestosterone beneoate In the same manner as Example 3, 11abenzoxy-lo-normethyltestosterone benzoate is prepared by reacting 11a-hydroxy-10-normethyltestosterone with more than about two molar equivalents of benzoyl chloride in pyridine.

In a similar manner, other esters of Ila-hydroxytestosterone and 11a-hydroxy-10-normethyltestosterone are prepared according to acylation procedures, as illustrated above or by reaction with ketene, ketenes of selected acids, selected acids, acid anhydrides, or acid chlorides, in an organic solvent such as pyridine or the like. Representative esters of lla-hydroxytestosterone and 1la-hydroxy-10-normethy1testosterone thus-prepared include one to eight carbon atom carboxylic acid acyloxy esters of saturated or unsaturated, aliphatic, carbocyclic, or cycloaliphatic, aryl, arylalkyl, alkaryl, mono, di or polycarboxylic acids which form ester groups such as, for example, formyloxy, acetoxy, propionyloxy, dimethylacetoxy, trimethylacetoxy, butyryloxy, hexanoyloxy, heptanoyloxy, octanoyloxy, benzoxy, phenylacetoxy, toluoyloxy, cyclopentylformyloxy, 8-cyclopentylpropionyloxy, :acrylyloxy, cyclohexylformyloxy, the half and di-esters of malonic, maleic, succinic, glutaric and adipic acids, and the like. The acids may also contain non-interfering substituents, such as mono or poly halo, chloro, bromo, hydroxy, methoxy, and the like if desired.

The lla-hydroxytestosterone, 11a-hydroxy-10 normethyltestosterone and their carboxylic acid esters are thermostable. These compounds have pharmacological activity per se and are useful as chemical intermediates for the production of other steroids which possess pharmacological utility. They demonstrate anesthetic and androgenic hormonal properties and function as ultraviolet screens. Saponification of Ila-acyloxytestosterone and oxidation of lloz-hYdIOXY- testosterone is productive of adrenosterone, 4- androstene-3,l1,17-trione. Similarly, saponification of 11a acyloxy 10 normethyltestosterone and oxidation of 1lot-hydroxy-l0-normethyltestosterone with chromium trioxide, CrOa, produce 10-normethyl-4-androstene-3,11,20-trione otherwise identified as 10 normethyladrenosterone having androgenic hormone activity.

It is to be understood that this invention is not to be limited to the exact details of operation or exact compounds shown and described as obvious modifications and equivalents will be apparent to valeryloxy,

one skilled in the art and the invention is therefore to be limited only by the scope of the appended claims.

We claim: 1. A compound represented by the graphical 5 formula:

wherein R is selected from the radicals consisting of hydrogen and methyl, and R is selected from the radicals consisting of hydrogen and hydrocarbon-carboxylic acyl radical containing from 20 one to eight carbon atoms, inclusively.

2. llu-hydroxytestosterone.

3. 1la-hydroxy-10-normethyltestosterone.

4. lla-acyloxytestosterone acylate of a hydrocarbon carboxylic acid having less than nine carbon atoms.

5. 1la-acyloxy-lo-normethyltestosterone acylate of a hydrocarbon carboxylic acid having less than nine carbon atoms.

6. lla-acetoxytestosterone acetate.

7. 11a-propionyloxytestosterone propiona-te.

8. Ila-(f3 cyclopentyl)propionyloxytestosterone B-cyclopentylpropionate.

9. lla-acyloxytestosterone acylate of an aliphatic hydrocarbon carboxylic acid having less than nine carbon atoms.

10. llu-acyloxytesterone acylate of a hydrocarbon carbocyclic carboxylic acid having less than nine carbon atoms.

No references cited. 

1. A COMPOUND REPRESENTED BY THE GRAPHICAL FORMULA: 